Fibrinop- and FibrinDegradation Products during Fibrinolytic Therapy” CHARCES w.FRANCIS& AND ABRAHAM KORNBERG Hemutdgy Unit, Dtpartmmt of Medicine UntpevJity of Roche~arSchool of Medicine Q Dentimy Rachesiq New Ymk 14642 The administration of a fibrinolytic agent accelerates dissolution of an occluding vascular thrombus by stimulating local fibrinolysis and plasmic degradation of its fibrin matrix. However, systemic activation of fibrinolysis also occurs, and this results in a variable degree of proteolytic degradation of plasma proteins including fibrinogen. Therefore, plasmic degradation products of both fibrinogen and fibrin circulate during fibrinolytic therapy. The ability to identify degradation products derived fi-om the thrombus separately fi-om those originating fkom plasma fibrinogen could be of significant value in monitoring the course of fibrinolytic therapy. Approaches to identification of fibrin-specific degradation products have been based on structural differences in degradation products of fibrinogen compared to those of fibrin. Plasmin action on fibrinogen results in initial cleavages of the carboxyl terminus of the Aar chains, and also release of the peptide Bola2 from the amino terminus of the BB chain, resulting in formation of fragment X. With further plasmin action, the coiledtoil regions linking the central and lateral domains are cleaved forming the intermediate fragment Y and terminal fragments D and E.1 The extent of fibrinogen degradation is variable during lytic therapy and is greater with streptokinase than with “fibrin-specific” agents such as tPA, but the extent of proteolysis is limited, and fragment X is the predominant derivative fbrmed.2 Plasmic degradation products of fibrin differ from those of fibrinogen owing to structural changes that accompany the conversion of fibrinogen to fibrin. One such change is the factor XIIIacatalyzed formation of isopeptide bonds between pairs of adjacent y-chains.3 Since the y-chain cross-link in stabilized fibrin renders this portion of the molecule resistant to plasmin, these bonds are retained in degradation products. A large number of macromolecular plasmic derivatives of cross-linked fibrin have been identified,’ the smallest of which is fragment DD, consisting of the D regions of two adjacent fibrin monomen cross-linked through y-chains.4-7 This work was supported in part by Grant No. HL-30616 from the National Heart, Lung and Blood Institute, National Institutes of Health,Bethesda, MD. Mdms comspondence to Charles W. Francis, M.D., Hematology Unit, P.O. Box 610, University of RDchester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642. Tel.: (716) 275-3761; FAX: (716) 473-4314.

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These differences in structure have been exploited in the development of several methods to identify circulating fibrin-specific degradation products as potential markers of clot lysis. These include electrophoretic methods to separate and identlfy fibrinogen and fibrin derivatives by size.8-12 Also, immunoassays based on fibrinspecific neo-epitopes associated with the 815-42 peptide, the y-chain cross-link,l3 and other sites14J5have been developed. Such assays have been evaluated in studies of patients undergoing fibrinolytic therapy fbr acute myocardial i n ! k C t i ~ n , deep l ~ ~ ~vein thrombosis (DVT),Zo and pulmonary embolism21 to determine if the appeatance of fibrin-specific degradation products would correlate with successfd dot dissolution. Generally, the results have shown little specificity, with elevations of both fibrinogenand fibrindegradation products in patients with or without vascular reperfusion. One explanation fbr these results is lack of specificity of the assays in identifying fibrindegradation products in the presence of large amounts of fibrinogendegradation pnxiucts. Another possibility is that the fibrin-degradation products derived from thrombus dissolution may be masked by lysis of fibrin from other sources.

SOLUBLE FIBRIN Fibrinolytic therapy is not “thrombus-specific,” and fibrin deposits at sites other than the symptomatic thrombus may be lysed, resulting in elevation of plasma fibrin degradation products. For example, patients treated fbr acute myocardial infirdon could have fibrin deposits in ulcerated atheromatous plaques, endocardial mural thrombi or coincident venous thrombi. Also, we have previously suggested that degradation of soluble fibrin in plasma may represent another important source of fibrindegradation products during fibrinolytic therapy22323 (FIG. 1). Hemostatic activation and thrombin fbrmation is primarily a localized process, but it may also have systemic effects including release of fibrinopeptide A from fibrinogen and circulation of soluble

Flbrinogon

lhrombln

Solubk Flbrin

I

-

Fibrin Ciol

trmnmglutmmlnmw

Crorrlinkrd Solubk Fibrin

Crorrlinkod Fibrin Clol

Fibrinogon

Solubk Flbrin

Fibrin Ciol

Flbrlnogen Dagndalion Produclr

Nwroadlnkod Flbrin Dagndollon Produeto

Crorllnkod Flbrin Dagrodollon Produtlr

FIGURE 1. Schematic illustration of the fixmation and plasmic degradation of fibrin.

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fibrin. The latter is heterogeneous in composition reflecting variable fibrinopeptide release, polymerization and cross-linking. Plasma soluble fibrin has been measured previously by several methods that have identified a mge of concentrations in normals. Values from 3 p g / m P to 27 pg/mP5 have been found using afEnity chromatography, and concentrationsfrom 25 &mP to as hq$ as 184 ~ / mhave l been reported using gel filtration chromatography.27 With a chromogenic assay based on stimulation of tissue-type plasminogen activator (@A) activity,a approximately 7 p g / d was found in normals. The wide range of concentrationsmay be ataibuted to &rent molecular species of soluble fibrin measured using these methods. Despite the differences, increases of between 3- and 10-fold have been identified in patients with thrombotic diseases including myocardial i n h t i o n , disseminated intravascular coagulation, stroke, and DVT.*31 Soluble fibrin in plasma may also be cross-linked. Using elecvophoretic techniques, we32 and have idendied low concentrations of cross-linked fibrin polymers in normal plasma and elevated concentrations in patients with thrombotic disease. In patients with acute myocardial infarction, the percentage of total fibrinogen present as dimer was fbund to be 4 f 1%,and this was significantly greater than the .8 f .l%in normal controls. Soluble fibrin polymers contain cross-linked y-chains implying that they are formed by the combined action of thrombin and fictor XIIIa. Several observations support the concept of such coordinated action of thrombin and fictor XI11 during activation of coagulation. During spontaneous dotting of plasma in vim, cleavage of fibrinopeptides and activation of fictor XI11 are closely related events3' The binding of thr~mbin38-3~ and fictor X I I P to fibrin may also explain the increase in thrombin-sadyzed fictor XIIIl by fibrin polymers in plasma.41 Earlier studies also provided indirect evidence for circulatingy-chain-cross-linked fibrin polymers by identifying y-chain dimers in extracts of plasma obtained from patients with thrombotic disease.8-10Recent evidence indicates that some degree of cross-linking between cy-chains or between a-and y-chains may also be present in plasma fibrin," indicating the possible action of tissue transglutaminase which prekrentially cross-links a- rather than y-chains.43 These polymers may also contain some fibrinogen as indicated by reaction with an antibody specific for fibrinopeptide A . 4 Since cross-linked fibrin polymers contain the plasmin-resistant y-chain cross-link, plasmic degradation yields derivatives containing cross-linked y-chain remnants, including fragment D-dimer. This can be demonstrated in vim using purified fibrinogen exposed to a low concentration of thrombin to increase the amount of cross-linked polymers. Plasmic degradation to terminal products yields both fi-agments D and DD. The amount of fragment DD in the digest, as determined by gel electrophoresis or by ELISA, is proportional to the amount of cross-linked polymer in the preparation before degradation.45 Cross-linked fibrin polymers in plasma can also degrade to fragment DD. Normal plasma incubated with a low concentration of thrombin demonstratesincreasing crosslinked polymers behre dot formation32 (FIG. 2). There is an increase not only in amount, but also in size, so that polymers of six or more units can be identified. Densitometric analysis indicated that the percent of cross-linked polymers in this experiment increased from 2.8%at baseline to 7.8%a t 20 min and 20.2%at 30 min, while clotting occurred at 36 min. As with the system using purified proteins, plasmic degradation generates fhgment DD in proportion to the amount of cross-linked polymer before degradation. Thus, the normal baseline plasma shown in FIGURE2 had a hint dimer band representing2.5%of the total by densitometric analysis; fragment DD was

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4’10 96 Polyacrylamide

2 % Agarose

-DD

-Tetramer -Trimer -Dimer

-D

-Monomer

1

2

3

1. 1

2

3

FIGURE 2. Formation and degradation of plasma cross-linked fibrin polymers. Left: Increasing cross-linked fibrin polymers in plasma after addition of thrombin. Normal citnted plasma was incubated with .01 units/ml thrombin and 10 m M calcium chloride at 37OC. Clot formation occurred at 36 min. Aliquots were taken prior to the addition of thrombin (hnc I) and at 20 and 30 minutes after thrombin addition and subjected to SDS 2% agarose electrophoresis followed by Western blotting with antifibrinogen antiserum. The amount and size of cross-linked fibrin polymers was increased following addition of thrombin and before clot formation. Right: Plasmic degradation of plasma containing increased amounts of cross-linked fibrin polymers formed by addition of thrombin. Aliquots of plasma comsponding to those in the left panel were incubated for one hour at 37OC with 25 pg/ml tissue plasminogen activator and 5 units/ml plasminogen. Aprotinin (500 units/ml) was then added to each digest, and an aliquot was subjected to SDS 410% gradient polyacrylamide gel electrophoresis followed by Western blotting with antifibrinogen antiserum. An increase in fragment DD is prcsent in +n of samples obtained at 20 minutes and 30 minutes and corresponding to samples in lanes 2 and 3 on the left panel. DD immunomctivity following in Pino plasmic digestion of the samples shown in lanes 1, 2 and 3 was 44 &ml, 160 pglml, 340 pglml, respectively. not visible by gel electrophoresisin the digest, and the D a r n e r immunoreactivity after digestion was 44 pg/ml. At 20 min, the fibrin polymers had increased to 7.8%, DD was easily identified by gel electrophoresis, and the DD concentration in the digest by ELISA was 160 &ml. At 30 min the amount of fibrin polymer befbre digestion was hrther increased, a prominent DD band was noted by gel electrophoresis, and the DD concentration by ELISA had increased 340 &ml. Similar results were found with patient samples. Thus, patients with acute myocardial inhction, stroke, or DVT showed increased fibrin polymers (FIG.3). Following plasmic degradation, a f%nt or no band corresponding to DD could be seen in normals, while the intensity of the band in patients was proportional to the amount of fibrin polymers seen before degradation. Similarly, the DD concentration in the plasmic digest as measured by ELISA reflected this relationship, with a concentration of 19 j@nl in normal plasma, and 88 &ml, 102 pg/ml and 174&ml in the patients with acute myocardial inhction, stroke, or DVT, respectively. Degradation in oiho of

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2% Agarose

4/10 % Polyacrylamide

DD

-

T etramer Trimer-

-

Dimer-

DMonomer-

1

2

3

4

1

2

3

4

FIGURE 3. Electrophoretic analysis of fibrinogen derivatives in plasma from normals and patients with thrombotic disease &re and afier plasrnic degradation. Left: SDS 2% w gel

clcctrophoresis of plasma from normals and patients bllowed by Western blotting with antifibrinogen antiserum. The sample in lane 1 was from a normal, lane 2 from a patient with acute myocardial i n h i o n , lane 3 from a patient with stroke, and lane 4 from a patient with DW. Cross-linked fibrin plymen in lanes 1-4 were: 18 &ml, 120 pg/ml, 160 Irglml and 280 Irglml, respectively as determined by dcnsitometric analysis and comparison with standards. Right: SDS 4-1096 polyacrylamide pcl electrophoresis of dqpts of the same plasma samples shown in thc left panel. Fngment DD is not visible in the digest of normal plasma but can be Seen in patient samples. DD immunoreactivity in samples 1-4 was: 19 Irglml, 88 Irglml, 102 pg/ml and 174 pglml, respectively.

fibrinogen and of fibrin polymers in plasma is incomplete after incubation with plasminogen activator, even at high concentration. Complete degradation to terminal h g ments E, D, and DD requires supplementation of the plasma with additional plasminogen, probably because of the high inhibitory capacity of plasma.

FIBRIN DEGRADATION PRODUCTS AND FIBRINOLYTIC THERAPY Elevated plasma concentrations of h g m e n t DD have been reported in patients with DVT14,4M8and pulmonary e m b o l i ~ m , prior ~ ? ~ ~to treatment, and this elevation reflects physiologic fibrinolysis. During fibrinolytic therapy, greater elevations in DD levels are fbund, with concentrations increasing 5-to 25-fold compared to pretreatment values.1621 However, despite the consistent increase, there has been poor correlation between the degree of thrombolysis and the elevations in D D during fibrinolytic therapy for acute myocardial infarction or DVT. For example, we measured plasma DD concentrations in patients with acute myocardial i n k t i o n treated with either intracoronary streptokinase or intravenous acylated p1asminogen:streptokinase activator complex WLE 1).% Elevated DD levels were found during treatment with both drugs. However, there was n o significant difference in D-dimer levels in patients who did or did not have angiographic reperfusion.

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TABLE 1. Plasma D-Dimer Concentrations in Patients with Myocardial I n h c t i o n Treated with Streptokinase or APSAC Therapy APSACa Streptokinase

Patients with Reperfusion

Patients with No Reperfusion

1096 f 88 (63) 587 f 48 (60)

875 f 67 (41) 634 f 95 (30)

848 f 55 (123)

TOTAL

773 f 57 (71)

Values are nglml, expressed as mean f SEM.Numbers in parentheses indicate the number of patients. APSAC was given intravenously as 30 U. Streptokinase was administered intracoronary at a dose of 160,OOO U over 60 min. (Data are from ref. 51.) a Acylated plasminogen: streptokinase activator complex.

Elevations in plasma fibrindegradation products during lytic therapy appear to reflect the intensity of the systemic pmteolytic state as shown by correlations between activator concentration, decrease in fibrinogen and elevation in fibrinogendegradation products.19 This is also suggested by results of gel electrophoretic analysis of plasma fibrinogen antigen during lytic therapy (FIG. 4).16All four patients with acute myocardial inhrction had increased plasma fibrin polymers before treatment with either intracoronary streptokinase or intravenous acylated p1asminogen:streptokinase activator complex. The second sample from patient 1 was obtained 37 rnin after coronary reperfusion at a time when the plasma fibrinogen had decreased to 24% of baseline,

I

2

3

4

TetramerTrimerDimer Monomer -

Before After Before After therapy reperfusion therapy reperfusion

Patients With Reper f usion

Before During Before During therapy therapy therapy therapy

Patients With

No R e p e r f u r i o n

FIGURE 4. Electrophoretic analysis of plasma samples from patients that received fibrinolytic therapy for acute myocardial i n k t i o n . Plasma samples obtained before and during or after fibrinolytic therapy were electrophorcsed on SDS 2% a&arose gels, and fibrinogen derivatives were identified after Western blotting. Prior to therapy, the plasma samples from all patients showed prominent fibrin polymer bands. Patients 1 and 2 had reperfusion demonstrated angiographically. Patient 1 developed a systemic lytic state with decrease in plasma fibrinogen, while no systemic lytic state developed in patient 2. No reperfusion occurred in patients 3 and 4. A systemic lytic state developed in patient 3 but not in patient 4. Fibrin polymers were degraded in both patients who developed a systemic lytic state (patients 1 and 3), but not in patients 2 and 4.

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and it shows heavy bands corresponding to fEagments Y and D as well as degradation of fibrin polymers. Electrophoresis of serum (not shown) confirmed the presence of fragments X, Y, and D and also showed high molecular weight cross-linked fibrindegradation products. Patient 2 also had reperfusion but, in contrast to patient 1, did not develop a systemic lytic state during therapy, and no degradation of fibrinogen o r fibrin polymers was noted. Patients 3 and 4 (FIG. 4, +t) did not experience reperfusion. However, the electrophoretic pattern of plasma from patient 3 was similar to that of patient 1 who did achieve reperfision. There was degradation of fibrinogen and of fibrin polymers during therapy with the appearance of both fibrinogenand crosslinked fibrin degradation products in serum. The pattern with patient 4, without reperfision, was similar to that of patient 2, with reperfusion. A systemic lytic state did not developand neither fibrinogen nor fibrin polymers degraded during therapy. Thus, electrophoretic analysis shows that fibrin polymers degrade during lytic therapy and that the degradation of both fibrinogen and cross-linked polymers correlates with the development of the systemic lytic state, but not with reperfision.

FIBRIN DEGRADATION PRODUCTS MAY DERIVE FROM SOLUBLE FIBRIN DURING FIBRINOLYTIC THERAPY The proposal that cross-linked fibrin-degradation products during lytic therapy may derive from cross-linked fibrin polymers is based on several considerations. Crosslinked fibrin polymers are bund in normal plasma and are increased in patients with thrombotic disease. Plasma cross-linked fibrin polymers contain the y-chain cross-link and degrade to fragment DD in pitro.. Further, electrophoretic analysis indicates that plasma cross-linked fibrin polymers degrade in piw during lytic therapy. Also, there is poor correlation between elevations in plasma DD levels during fibrinolytic therapy and the Occurrence of thrombolysis. Another important consideration is that the size of many thrombi limits the potential increase in plasma fibrindegradation products resulting from their dissolution. For example, a coronary artery thrombus with a diameter of 3 mm and a length of 2 cm would have a volume of .14 ml. Assuming that this thrombus contained no red cells and had a fibrin concentration Similar to that in plasma (3 mg/ml), such a thrombus would contain approximately 420 pg of cross-linked fibrin. Complete lysis would generate 250 pg of fragment DD, which would elevate the plasma DD concentration by 83 ng/ml after dilution in a plasma volume of 3000 ml. By contrast, the amount of DD that could potentially derive from degradation of cross-linked fibrin polymers is much greater. Considering that 1%of plasma fibrinogen in normals (3000 pglrnl) is cross-linked dimer, then the plasma concentration of dimer is approximately 30 pg/ml. Since fragment DD represents 30% of a y-chain cross-linked fibrin dimer, complete degradation would generate 9000 ng/ml. Therehre, during fibrinolytic therapy, the contribution to circulating DD from lysis of a coronary clot could easily be masked by the larger amount of DD potentially derived from cross-linked fibrin polymers. The amount of fibrin in a deep venous thrombus is much larger than that in a coronary thrombus, and, consequently, there is a better chance of detecting thrombusderived fibrin degradation p d u c t s during lytic therapy fbr DVT. Therebre, we examined a cohort of patients who received therapy with tissue plasminogen activator fbr DVT, examining the possibility that the elevation in fibrin-specific degradation prod-

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ucts would correlate with clot lysis.23 We hypothesized that a significant correlation would be seen only if the contribution from degradation of plasma cross-linked fibrin polymers was considered in the analysis. This was done by incubating plasma samples obtained from each patient before lytic therapy with plasminogen activator in pitra to estimate the amount of D-dimer immunoreactivity potentially derived from fibrin in the plasma before treatment. We found that incubation of plasma in Pino with a hq$ concentration of activator, but without supplemental plasminogen, resulted in incomplete degradation of fibrinogen and cross-linked fibrin polymers, and this was similar in extent to that seen during fibrinolytic therapy. D-dimer concentration was measured in several samples obtained bebre, during and after fibrinolytic therapy for DVT. There was typically a marked increase after initiation of therapy to a high level which was sustained until therapy was terminated, and then the concentration decreased. These results were plotted and interpreted in relation to the concentration of D-dimer derived following in piho incubation of pretreatment plasma with activator. FIGURE5 shows this analysis. At baseline, there was a low plasma concentration of DD which increased rapidly after administration of fibrinolytic therapy, stayed at a high level and then declined after treatment was stopped. Two hypothetical cases are illustrated. If incubation of the pretreatment plasma with activator in resulted in a DD concentration represented by A, then the area under this level, represented by 1, would approximate the contribution from plasma, and the area above, (2 + 3), would be that from the thrombus. In contrast, if there was a higher concentration of soluble fibrin, then the in piho digest of the pretreatment plasma would have a higher DD concentration, corresponding to B in FIGURE5. In this case, most circulating DD, including areas 1 + 2, could derive from the plasma source and only the small area, labeled 3, from the thrombus. Plasma DD concentrations were analyzed in this way in a group of 13 patients with DVT who were enrolled in an open, rising-dose, safety and dose-ranging study of recombinant tissue type plasminogen a~tivator.~' Venous blood samples were obtained before and at 1, 2, 6, 12, 36, 48 and 72 h after the start of therapy. Ascending venography was performed before and at 6-16 h after the termination of the activator infusion. Clot lysis was quantitated by comparison of the pre-treatment and postL y l i c Therapy FIGURE 5. Analysis of cross-linked z fibrin degradation products during fibrinolytic therapy. A low concentrarr tion of fibrin degradation products is present before administration of lytic therapy. The concentration increases rapidly after institution of therapy, rev mains elevated, and then declines after A 0 therapy is stopped. Two hypothetical 0 cases with the same elevation of fibrin TIME degradation products are shown. In A, addition ofplasminogcn activator to the baseline, pretreatment plasma sample resulted in generation of a low level of D-dimer i m m u n e reactivity. This would be interpreted to mean that the amount of D-dimer rcpresented by area 1 was derived in such a patient from degradation of plasma fibrin polymers while the area r e g resented 2 + 3 originated from clot lysis. In the case represented by B, addition of plasminogcn activator to the pretreatment plasma sample generated a much higher concentration of D-dimer immunoreactivity. Thercbre, the amount of D-dimer represented by areas 1 + 2 derived from plasma SOUKCS while only that rcpresented by 3 originated from dot lysis.

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treatment venograms using a modification of the technique of Marder et al.52 in which the volume of lysed clot was calculated by multiplying the cross-sectional area of the reperfused vessel by its length. Cross-linked fibrin degradation products were measured with an ELISA (Dimer Test, American Diagnostics, Greenwich, cr) using plates pre-coated with monoclonal DD/3B6 and a secondary, enzyme-linked antibody reactive with both fibrinogen- and fibrindegradation products. Pretreatment DD concentrations were increased in patients with venous thrombosis (1074 f 252 ng/ml) compared to normals (75 f 3 ng/ml). DD levels rose after initiation of therapy to a peak of 10 333 f 1004 n g / d at 6 h and then declined to pretreatment levels at 72 h. After therapy, repeat venograms showed lysis of 3 4 5 ml in 8 patients and no lysis in 5 patients. DD concentrations were compared with the venographic change for each patient (FIG.6). No correlation was found between measured peak DD concentration and clot lysis, but a moderate correlation (Y .62) was found between the volume of lysed clot and the peak DD value after correction for the contribution tiom pretreatment plasma fibrin polymers. Similarly, the correlation demonstrated between the measured time-integrated DD and clot lysis (I = .47) was improved after correction for DD derived from soluble fibrin (Y .97). A similar analysis was perfbrmed on samples obtained from 27 patients with myocardial i n k t i o n who received fibrinolytic therapy.23 TIM1 grade 2 or 3 patency was achieved in 21 of the 27, while 6 had persistent arterial occlusion. Post-treatment plasma DD levels were elevated to the same extent in patients with or without reperfusion, and the corrected post-treatment DD concentration was -120 f 134 ng/ml in patients with reperfusion and -213 f 413 ng/ml in patients without reperfusion. This indicated that all of the plasma DD after therapy could be attributed to plasma sources, and that there was no significant increase in DD resulting from lysis of the small coronary artery thrombus.

-

-

DISCUSSION

These finding support the hypothesis that fibrin degradation products during thrombolytic therapy derive from both the thrombus and from degradation of soluble fibrin. The DD concentration in the baseline plasma sample after in vim incubation with rtPA represents the sum of DD present in the plasma before treatment plus that produced by degradation of soluble fibrin (FIG. 5). Higher concentrations during therapy therefore result from lysis of fibrin in thrombi. This was observed with successful treatment of DVT when good correlation between the degree of elevation of plasma DD and the extent of lysis was observed (FIG.6). While this method corrects for the contribution of soluble fibrin to elevated plasma DD concentrations during fibrinolytictherapy, there may be additional intravascular or extravascular sites of fibrin deposition. However, the finding of a strong correlation between the DD levels and quantitative venous clot lysis after correction for soluble fibrin degradation suggests that other fibrin deposits did not contribute significantly to the result. Further, the net corrected DD value was close to 0 in patients treated for myocardial i n k t i o n , and this argues against the presence of unknown, non-coronary thrombi in these patients. An alternative explanation for high levels of fibrindegradation products during fibrinolytic therapy and the lack of correlation with thrombolysis is nonspecificity of the immunologicassay. Thus, the assay for D-dimer that we have used employs a fibrin-

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.

B .

P 0,

D

s

r = 0.03

E

r = 0.62

2

2

0 2ooooc

E

5

15000-

2

8 U 3 I-:;.

0

1

2 a

Lu

U

.

0

0 0

0

5000-

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!2

0 ,

I

I

I

I

LYSED CLOT (ml)

I

= 0.47

Q

E

20

E

z

’.B 8

0

00

10

20

30

40

50

0

10

20

30

40

50

LYSED CLOT (ml)

FIGURE 6. Scatterplot of correlation of D-dimer concentrations with clot lysis in patients with deep vein thrombosis. The volume of clot lysed during therapy was estimated by comparison of pretreatment and post-treatment venograms. Correlations between the volume ofclot lysed with measured and corrected peak concentrations (top A) and with measured and time-integrated Ddimer concentrations (bottom B)att presented. The “corrected peak concentration” represented the peak D-dimer concentration measured in patient plasma minus the concentration in the pretreatment plasma sample following incubation with plasminogen activator. The “corrected integrated D-dimer” was obtained by subtracting the a m below the line representing the contribution from soluble fibrin lysis from the total integrated m a of D-dimer concentration versus time (see FIG.5). (Figure from ref. 23, used by permission of the American Heart Association, Inc.)

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ANNALS NEW Y O U ACADEMY OF SCIENCES

specific antibody (3B6)as the capture antibody, but the secondary antibody reacts with both fibrinogen- and fibrindegradation products.14 It has been suggested that fibrinogendegradation products may associate noncovalently with hgment DD in the assay and thereby lead to a spuriously elevated ~ r s u l tThis . ~ ~ possibility is supported by reports using assays employing two fibrin-specific monoclonal antibodies. These showed no increase in immunoreactivity after plasma was incubated in PihD with plasminogen activators.19-53Such assays may be more specific h r fibrindegradation products, but their reactivity with mss-linked fibrindegradation products larger than DD, which may be present after digestion is not known, and clinical studies evaluating their value in predicting thrombolysis are not available. However, prior reports have hund no reaction of the assay using antibodies 3B6 and 4D2 with fragment D at concentrations up to 200 d m l , I 4 3 which fir exceeds the concentration of fragment D in digest samples after dilution. Several lines of evidence indicate that degradation of soluble fibrin contributes to the elevation in fibrindegradation products during thrombolytic therapy. First, soluble fibrin has been identified in plasma by several methods and increased concentrations measured in patients with thrombotic disease. This soluble fibrin would be expected to degrade at least to the same extent as fibrinogen hllowing administration of plasminogen activator. Second, degradation of cross-linked fibrin polymers in piw has been observed during fibrinolytic therapy (FIG.4). Third, incubation of plasma with a low concentration of thrombin in piho increases cross-linked fibrin polymers, and parallel increases occur in D-dimer immunoreactivity and in fragment DD found by electrophoretic analysis after plasmic degradation (FIG. 3). Fourth, fragment DD is fbrmed in plasma of patients followingfibrinolyticactivation in vim, and its concentration by electrophoretic analysis or immunoassay correlates with the plasma content of cross-linked polymers behre degradation (FIG. 4). In conclusion, administration of plasminogen activator results in a decrease in clottable fibrinogen and a corresponding increase in degradation products of fibrinogen. The degree of elevation of fibrinogendegradation products and their extent of degradation is related to the intensity of the systemic lytic state induced by therapy. Fibrinolytic therapy also results in the circulation of fibrindegradation products which may derive from multiple sources. One source is lysis of vascular thrombi, and this will elevate fibrindegradation products in proportion to the amount of fibrin lysed. However, soluble fibrin in plasma will also be degraded and contribute to the elevation in fibrin-degradationproducts. The total elevation will reflect the sum of both processes. Measurement of fibrin-specific degradation products during thrombolytic therapy may be a useful monitor of clot dissolution after appropriate adjustment of concentrations b r the contribution from degradation of soluble fibrin.

REFERENCES 1. MARDER,~. J. & A . Z . BUDZYNSKI.1974.Thestructureofthefibrincgendegradationproducu. Prog. Thmmb. Hemostasis 2: 141. 2. OWEN,J., K. D. FRIEDMAN,B. A. GROSSMAN, C. WXLKINS, A. D. BERKE& E.R POWERS. 1987. Quantitation of fi-agment X fbrmation during thrombolytic therapy with streptokinase and tissue plasrninogen activator. J. Clin. Invest. 79: 1642-1647. 3. CHEN,R & R F. -LITTLE. 1971. y-y cross-linking sites in human and bovine fibrin. Biochemistry 10: 44864491. 4. FRANC IS,^. W . , V . J. W E R & G . H. B m w . 1980. Plasmicdegradation ofcrosslinked

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5. 6. 7.

8. 9.

321

fibrin. Characterization of new macromolecular soluble complexes and a model of their structure. J. Clin. Invest. 66: 1033-1043. GAFFNEY, P. J. & M. BRASHER.1973. Subunit structure of the plasmin-induced degradation products of crosslinked fibrin. Biochem. Biophys. Acta 295: 308-311. Przzo,S. V., L. M. TAYLOR,JR., M. L. SCHWARTZ,R L. HILL& P. A. MCKEE. 1973. Subunit StIUCNre of fragment D from fibrinogen and crosslinked fibrin. J. Biol. Chem. 248: 4584-4590. KOPEC,M., E. TEISSEYRE, G. DUDEK-WOJCIECHOWSKA, M. KLOCZEWIAK, A. PANKIEWICZ & Z. S. LATALLO.1973. Studies on the “Double D” m e n t from stabilized bovine fibrin. Thromb. Res. 2: 283-291. LANE,D. A., F. E. PRESTON, M. E. VANROSS& V. V. KAKKAB. 1978. Characterization of serum fibrinogen and fibrin fi-agrnents produced during disseminated intramcdar coagulation. Br. J. Haematol. 40: 609-615. FRANCIS, C. W ., V. J. MARDER& S. E. hlARTIN. 1979. Detection of circulatingcrosslinked fibrin derivatives in a heat extractionSDS gradient gel electrophoretic technique. Blood

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